Non-invasive ocular biomarkers for early diagnosis of diseases

ABSTRACT

Materials and methods are disclosed for screening advanced glycation end-products from mammalian ocular lens proteins to quantify early biomarkers for the diagnosis of diseases such as diabetes mellitus and to prevent related complications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/557,727, filed Sep. 12, 2017, which is a U.S. nationalization under35 U.S.C. § 371 of International Application No. PCT/US2016/028976,filed Apr. 22, 2016, which claims the benefit of priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 62/151234, filedApr. 22, 2015. The disclosures set forth in the referenced applicationsare incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 21, 2016, isnamed 253275_SEQ_ST25.txt and is 4,096 bytes in size.

BACKGROUND

Various human diseases and conditions have ocular components. The humaneye lens is a transparent, biconvex structure that helps to refractlight to be focused on the retinal surface. The change in curvaturehelps in adjusting the focal distance of the eye so that it can focus onobjects at different distances. This adjustment of the lens is calledaccommodation. Presbyopia is a common ocular condition observed inpatients above 50 years and is characterized by loss of flexibility ofthe crystalline eye lens and in turn, accommodation. Cataract is theleading cause of blindness, affecting 40 million people worldwide. It isa multifactorial ocular disease caused by genetics, age, andenvironment. There are reports that glycation gradually damages the lensby causing aggregation of the lens proteins.

Diabetes Mellitus

Diabetes mellitus is an endocrine metabolic disorder characterized byhigh blood sugar levels which give rise to complications in the eye,kidneys and the brain. Diabetes triggers the development of oculardiseases, for example, diabetic retinopathy, glaucoma and cataractswhich are the leading cause of blindness around the world. The mostcommon method for the diagnosis of diabetes involves measuring the bloodsugar levels in the body. One major disadvantage of this method is thatblood sugar levels fluctuate which contributes to false negativeresults. This leads to delay in treatment, eventually causing permanentdamage to the organs. Therefore, diagnosis of diabetes at an early stageis very crucial. Additional or alternative diagnostic tests would bebeneficial.

Diabetes arises due to inadequate insulin production, or because thebody's cells are non-responsive to insulin, or both. Globally, about 382million people have diabetes, of which around 46% remain undiagnosed.Diabetes mellitus starts a vicious cycle of diseases affecting theheart, kidneys, eyes and the nervous system. The high blood sugar levelshave been observed to cause damage to small blood vessels in theseorgans by destroying their structure. The total estimated cost ofdiagnosed diabetes in 2012 is $245 billion which increased to $548billion in 2013. Overall, the number of diabetics as well as theexpenditure including direct and indirect costs have been increasingdrastically.

Glycated Proteins

In addition to blood sugar levels, there are several other methods usedfor the diagnosis of diabetes. Measurement of glycated proteins,primarily glycated hemoglobin (HbA1c or A1C), has been widely used forroutine long-term monitoring of glucose control and as a measure of riskfor the development of diabetes complications. The A1C test measuresaverage blood glucose for the past 2 to 3 months and, if the values aregreater than or equal to 6.5%, the person is considered diabetic. TheFasting Plasma Glucose (FPG) and Oral Glucose Tolerance (OGT) testsmeasure the blood sugar levels after fasting and having a sweet drinkrespectively. Values greater than or equal to 126 mg/dl and 200 mg/dlare considered diabetic in FPG and OGT respectively. (Table 7)

Although, these tests give accurate results in diabetic patients, theyhave their exceptions. The blood glucose levels in the body fluctuatedepending on the meals, exercise, sickness, and stress. It has also beenshown that different diagnostic tests might give varying results and notagree with one another. The glycation of hemoglobin occurs at severalamino acid residues and, as a result, several adducts of hemoglobin A(HbA) and various sugars are formed by the non-enzymaticpost-translational glycation process. This process involves theformation of a labile Schiff base intermediate followed by the Amadorirearrangement. The reaction is slow, irreparable, and the reaction ratedepends on the ambient glucose concentration. Also, these tests do nottake into consideration that the proteins, especially in thevasculature, have rapid turnover and hence are not always the same overtime.

HbA1c has been recommended as an accurate and precise marker fordiabetes based on advances in instrumentation and standardization. Thetheory behind the A1C test is that red blood cells live an average ofthree months. So, if the amount of glycated hemoglobin is measured,results will give an idea of glycation that occurred over the last 3months. But, research has shown that the lifetime of red blood cells ofdiabetics is comparatively shorter than that of non-diabetics. Thismeans that the hemoglobin turnover is faster in case of diabetics, andtherefore is a major disadvantage for diagnosing the patients in theirearly stages. Also, it has been reported that people with hemoglobinvariants, for example, HbC and HbS have shown false negatives. A falseelevated A1C level has been observed in certain clinical situations thataffect RBC life span with iron deficiency anemia, high alcoholconsumption and hypertriglyceridemia. Other cases which have shown falseresults include patients with kidney failure and liver disease.

Ocular Proteins

Three major proteins called α-, β- and γ-crystallins are found in theeye lens. The structure, biochemical and physiological properties aswell as functionalities of these crystallins have been reported. Themonomeric γ-crystallins are globular and the smallest with a molecularweight of about 20 kDa. In the case of β-crystallins, their subunitsform oligomers with low molecular weight species (β_(L)-60 kDa) and highmolecular weight species (β_(H)-160 kDa). α-crystallin is the mostabundant and largest of the lens proteins (˜18 nm in diameter)consisting anywhere between 30-40 subunits with molecular weight rangingfrom 800-1200 kDa.

Due to a very low protein turnover, crystallins are considered to besome of the longest-lived proteins in the human body. Because of thelong half-life, α-crystallin is prone to irreversible modificationsleading to changes in structure and function. The most commonly observedpost-translational modifications include photo-oxidization, deamidation,racemization, phosphorylation, acetylation, glycation and age-dependenttruncation. Post-translational modifications alter protein-proteininteractions and subsequently destabilize and reduce the solubility ofnative crystallins.

Alpha-Crystallins

The eye lens is avascular and constitutes a dense matrix of closelypacked proteins. α-crystallin is a major water soluble small heat shockprotein (˜45%) found in the eye lens. It is isolated from vertebrate eyelens as a polydisperse, hetero-oligomeric complex of approximately800-1200 kDa, consisting of 35-40 subunits. It is made up of twodistinct sub-units—A and B in the ratio of 3:1 respectively. It has achaperone function, protecting other proteins and crystallins fromthermal aggregation. This in turn helps in maintaining the transparencyof the eye lens. Recently, it has been observed that α-crystallinsub-units are not restricted to the eye lens, but also are expressed inother non-lenticular tissues like retina, heart, brain and kidneys.While αA is mostly restricted to the lens and retina, it has beenreported that αB subunit is expressed ubiquitously in cells undergoingstress.

In its native form, α-crystallin consists of two homologous subunitsshowing 55% sequence similarity in a ratio of 3:1—αA and αB, with 173and 175 amino acid residues, respectively. The molecular weight of thesetwo subunits is approximately 20 kDa. αA crystallin is confined to thelens with a small amount in the retina, spleen and thymus. αB crystallinis ubiquitously present in the lens, retina and the heart abundantly,and is expressed under stress and pathological conditions in the spinalcord, muscles, brain and the kidneys. There are reports thatα-crystallins act as anti-apototic regulators and prevent apoptosisunder stress conditions, thereby protecting the tissues from damage

A recent study has detailed how in concentrated suspensions of alphacrystallin, inter-particle correlations are well described by thestructure factor for a hard sphere fluid.

The chaperone function of α-crystallin has been reported to preventthermal aggregation of other proteins. Over a period of time,α-crystallin undergoes irreversible post-translational modifications ofwhich non-enzymatic glycation is prominent especially in aging anddiabetes. As a result, the protein slowly starts losing its chaperoneability and starts to aggregate. Glycation also leads to loss ofanti-apoptotic activity of alpha crystallin.

Although α-crystallin has been studied extensively, the quaternarystructure of the native protein has not been elucidated. As a result,the location of protein modifications which are a part of diseasepathology are not resolved. As the lens is avascular and has noturnover, the modifications that occur in the lens alpha crystallin dueto non-enzymatic glycation are permanent. Reducing sugars react withbasic amino acids of proteins to form Schiff's bases which undergorearrangement to Amadori products and finally form advanced glycationend-products (AGEs). These AGEs lead to loss of protein integrity,increase hydrophobicity and play an important role in proteindenaturation.

Protein denaturation is usually associated with the formation ofaggregates. Protein precipitation and aggregation involves the growth oflarge sized particles and hence is an optimum method for biophysicalcharacterization based on particle size. Light scatteringcharacteristics of protein aggregation of crystallin glycation effectson the protein and its role in the decrease of lens flexibility(presbyopia) as well as the formation of cataracts need to bedetermined.

An important biomarker for diabetes related diseases is the formation ofadvanced glycation endproducts (AGEs). AGEs are formed due tonon-enzymatic glycation of the proteins on exposure to open chain sugarsand dicarbonyl intermediates. Hyperglycemic conditions, oxidative andthermal stress lead to the formation of Schiff's bases with basic aminoacids like lysine and arginine. Further, Amadori products are formed dueto rearrangement of the Schiff's bases when highly reactive carbonylintermediates accumulate and attack the amino and guanidine groups onthe proteins. Unlike hemoglobin and albumin, the heat shock proteinshave long half-lives and very low turnover. As a result, theaccumulation of glycation products over a long period of time is likelyof quantitative significance.

Methylglyoxal (MGO) is a glycating agent that is generatednon-enzymatically from the oxidation and spontaneous dismutation ofintermediates in the glycolysis pathway or enzymatic oxidation reactioncatalyzed by peroxidases. MGO is reported to be toxic and to interferewith cellular mechanisms. It has been reported to impair functions ofmitochondria and also produce reactive oxygen species. Another source ofthis dicarbonyl reactive intermediate in the body is deficiency oftriose phosphate isomerase leading to elevated dihydroxyacetonephosphate(DHAP) levels observed in congenital hemolytic anemia and otherneurodegenerative diseases. DHAP spontaneously disintegrates tomethylglyoxal which acts as a strong agent in the formation of advancedglycation end products (AGEs). Because MGO reacts rapidly with theproteins, modification by MGO is a good in vitro model for investigatingthe long term effects of glycation on heat shock proteins. (FIG. 17)

SUMMARY

By screening the changes in the ocular lens proteins and, consequently,the intact lens, non-invasive spectroscopic biomarkers were identifiedfor the early diagnosis of diabetes mellitus. The appearance of thesebiomarkers will indicate commencing a treatment regimen, therebypreventing further complications.

Steady state and time resolved fluorescence measurements were used tostudy the spectroscopic changes in a-crystallin with increase in time ofglycation, in intact lenses from diabetic and nondiabetic donors. Anoninvasive diagnostic tool for early detection of diabetes mellitus isdisclosed.

A method for diagnosing a disease by non-invasive scanning of the ocularcells and tissue, primarily from the lens, includes:

-   -   (a) detecting fluorophores that formed in the ocular cells and        tissue by steady state fluorescence and recording results;    -   (b) using the results of the steady state fluorescence detection        of (a) to determine the emission maxima;    -   (c) quantifying the fluorophores using time resolved        fluorescence for determining the fluorescence lifetimes of the        fluorophores; and    -   (d) using the fluorescence lifetimes to distinguish between        normal and pathological ocular cells and tissue.

Ocular disorders include those related to diabetes mellitus.Non-enzymatic glycation-induced structural damage in alpha-crystallinwas investigated using biophysical and spectroscopic characterization.Non-enzymatic glycation of proteins for example by MGO, leads toformulation of AGEs and aggregation. Correlations between thestructured, molecular and spectroscopic changes in the glycating agentmethylglyoxal (MGO) as it slowly denatures due to aggregation induced bynon-enzymatic glycation, is a model for the effects of aging anddiabetes.

Aspects of the disclosure also relate to a method for diagnosing anocular disorder or disease by screening the spectroscopic changes withaging and pathological conditions in the eye where the tissue includes acombination of cornea, lens, vitreous and the retina.

Fluorophores are formed on the small heat shock lens proteins, forexample, alpha crystallin. The fluorophores include specificallyadvanced glycation end-products (AGEs) when measured in vitro. Whenmeasured in vivo, the fluorophores include a combination of advancedglycation end products formed on macromolecules in the eye. The productsinclude A2E, lipofuscin, FAD, NADH and any other fluorophores formed dueto aging and/or pathological conditions in the eye.

A system for diagnosing an ocular disease includes quantifying thespectroscopic changes in the visible spectrum between 401-600 nm basedon fluorescence lifetimes with aging and pathological conditions, in thesmall heat shock protein, alpha crystallin and its sub-units.

An apparatus for detecting fluorophores in an ocular cell or tissueincludes means for focusing an excitation light beam on the ocular cellor tissue; means for detecting a fluorescent light emitted from the cellor tissue in a one or more wavebands, wherein the detection means isoperable to produce an intensity of fluorescence from the cell ortissue; means for calculating the intensity of fluorescence for eachwaveband, wherein the calculation means discerns the detection of thefluorophore in the cell or tissue; means for measuring the fluorescencedecay with time after a short excitation pulse from the cell or tissuein the specific waveband; means for calculating the fluorescencelifetimes from the fluorescence decay curves using global 1-4 threeexponential fitting analysis.

The fitting analysis is assessed based on good autocorrelation functionaround zero, weighted number of residuals randomly distributed between+3 and −3, reduced chi square values between 0.9-1.1 and Durbin-Watsonparameters of greater than or equal to 1.6, 1.7 and 1.8 for one, two andthree exponential decay respectively.

An in vitro diagnosis for distinguishing the normal ocular tissue frompathological tissue is based on the fluorescence intensity and lifetimemeasurements at specific excitation wavelengths between 280-600 nm, andthe damage to the proteins in the tissue are confirmed based on X-rayscattering.

The disclosed methods alone or in combination are useful in artificialintelligence, diagnostic, biotechnology, veterinary and forensicapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a diagrammatic representation of a prototype instrument forthe early diagnosis of diabetes using measurements obtained frommammalian eyes.

FIG. 2. Steady state fluorescence spectra of α-crystallin with increasein time of glycation at an excitation wavelength of 340 nm.

FIG. 3. Steady state fluorescence spectra of α crystallin with increasein time of glycation at an excitation wavelength of 435 nm.

FIG. 4A. Comparison of steady state fluorescence measurements at anexcitation wavelength of 340 nm between non-diabetic (56 and 77 yr old)and diabetic (59 and 70 yr old) human donor lenses. FIG. 4B. In theinset, comparison of steady state fluorescence measurements at anexcitation wavelength of 340 nm from non-diabetic human lenses fromdonors—20, 56 and 77 year old at the time of death can be observed.(Emission maxima 420-430 nm).

FIG. 5A. Comparison of steady state fluorescence measurements at anexcitation wavelength of 435 nm between non-diabetic (56 and 77 yr old)and diabetic (59 and 70 yr old) human donor lenses. FIG. 5B. In theinset, comparison of steady state fluorescence measurements at anexcitation wavelength of 435 nm from non-diabetic human lenses fromdonors—20, 56 and 77 year old at the time of death can be observed.(Emission maximum 500 nm)

FIG. 6. Increase in hydrodynamic diameter of α-crystallin afterincubating with 10 μM methyl glyoxal over a period of 9 hr at 25° C.

FIG. 7. Small angle X-ray scattering data to measure the inter-particledistances of unmodified and glycated α-crystallin.

FIG. 8. Change in surface hydrophobicity of the native α-crystallin and10 μM methyl glyoxal modified α-crystallin over a period of 9 hr at 25°C.

FIG. 9. Change in tryptophan fluorescence of α-crystallin afterincubating with 10 μM methyl glyoxal over a period of 9 hr at 25° C.

FIGS. 10-12. are photographic comparisons of steady state fluorescencehotspots in matched diabetic and controlled samples. FIG. 10A. is from a18 year type 1 diabetic, FIG. 10B. is from 20 year old control.

FIG. 11A. is from a 42 year old diabetic, FIG. 11B. is from a 42 yearold control.

FIG. 12A. is from a 56 year old diabetic with retinopathy, FIG. 12B. isfrom a 56 year old control.

FIG. 13. shows results of fluorescent lifetimes.

FIG. 14. shows spectral hotspots 14A. in a non-diabetic 46 year olddonor, FIG. 14B. in a diabetic 49 year old donor.

FIG. 15. is graphical abstract of diabetic versus non-diabetic tissues,FIG. 15A. is from a 46 year old non-diabetic donor lens; FIG. 15B. timeresolved fluorescence graph distinguishing non-diabetic from diabeticlens tissue; FIG. 15C. is from a 42 year old diabetic donor lens.

FIG. 16. Mechanism of chaperone action of α-crystallin.

FIG. 17. Non-enzymatic glycation of proteins by simple sugars anddicarbonyl intermediates. (http://www.edb.hr/dialogia.01no2-2.html)

FIG. 18. Change in UV-Vis spectrum of α-crystallin after incubating with0.05M glycolaldehyde at 25° C. over a period of 5 weeks.

FIG. 19. Change in UV-Vis absorbance of α-crystallin after incubatingwith 10 μM methyl glyoxal at 25° C. over a period of 9 hr.

FIG. 20. Change in hydrodynamic diameter of α-crystallin afterincubating with 1M glucose, 1M sucrose and 0.05M glycolaldehyde at 25°C. over a period of 5 weeks.

FIG. 21. Change in translational diffusion coefficients of α-crystallinafter incubating with 1M glucose, 1M sucrose and 0.05M glycolaldehyde at25° C. over a period of 5 weeks.

FIG. 22. Change in translational diffusion coefficients (Diffusivity) ofα-crystallin after incubating with 10 μM methyl glyoxal at 25° C. over aperiod of 9 hr.

FIG. 23. Change in relaxation times of α-crystallin after incubatingwith 1M glucose, 1M sucrose and 0.05M glycolaldehyde at 25° C. over aperiod of 5 weeks.

FIG. 24. Change in relaxation times of α-crystallin after incubatingwith 10 μM methyl glyoxal at 25° C. over a period of 9 hr.

FIG. 25. Static Light Scattering data of 10 μM methyl glyoxal modifiedα-crystallin at 25° C. showing an increase in molecular mass with timeof glycation.

FIG. 26. Small angle X-ray scattering data for measuring inter-particledistances in native state α-crystallin.

FIG. 27. Small angle X-ray scattering data for measuring inter-particledistances in glycated α-crystallin.

FIG. 28. Change in surface hydrophobicity of the native α-crystallin and10 μM methyl glyoxal modified α-crystallin over a period of 9 hr at 25°C.

FIG. 29. Changes in the amount of water soluble and insoluble fractionsfrom the control calf lens (0 hr) and 10 μM methyl glyoxal modified calflens over a period of 9 hr at 25° C.

FIG. 30. Change in refractive index due to formation of high molecularweight aggregates and advanced glycation endproducts after glycatingα-crystallin using 10 μM methyl glyoxal over a period of 9 hr at 25° C.

FIG. 31. Change in secondary structure of α-crystallin with increase intime of glycation using 10 μM methyl glyoxal over a period of 9 hr at25° C.

FIG. 32. Change in tryptophan fluorescence of α-crystallin afterincubating with 10 μM methyl glyoxal over a period of 9 hr at 25° C.

FIG. 33. Comparison of steady state fluorescence hotspots between FIG.33B. 64 yr old non-diabetic and FIG. 33A. 67 yr old with cataracts.

FIG. 34. Steady state fluorescence spectra of α-crystallin with increasein time of glycation at an excitation wavelength of 340 nm.

FIG. 35. Steady state fluorescence spectra of α-crystallin with increasein time of glycation at an excitation wavelength of 370 nm.

FIG. 36. Steady state fluorescence spectra of alpha crystallin withincrease in time of glycation at an excitation wavelength of 435 nm.

FIG. 37. Small angle X-ray scattering data to measure the inter-particledistances of unmodified and glycated α-crystallin.

FIG. 38A-B. Positions of several residues in α-crystallin A model andα-crystallin B model.

DETAILED DESCRIPTION

Physical and chemical biomarkers were sought for the glycolyticmodifications in the ocular lens in order to develop a diagnostic toolfor the early diagnosis of eye diseases related to other disorders, e.g.diabetes.

The eye lens doesn't have any protein turnover, and therefore is anideal tissue to study the early stage glycation due to hyperglycemicconditions. By screening for the formation of AGEs and quantifying theresulting physicochemical changes, the development of diabetes may bediagnosed at a very early stage. This in turn, will help in initiatingthe treatment/precautionary measures from an early stage and prevent theprogression of the diseases like diabetic retinopathy and loss ofvision.

Lens proteins as well as intact lenses were glycated and the formationof advanced glycation endproducts (AGEs) were characterized using steadystate fluorescence and time resolved single photon counting. Thebiophysical changes in the protein were studied using Dynamic and StaticLight Scattering, Small Angle X-ray Scattering to measure the change inscattering intensities.

From the steady state fluorescence measurements, a resonance energytransfer was observed between tryptophan and AGEs. Also, with increasein time of glycation, the AGEs absorbed at wavelengths longer than 370nm. The fluorescence lifetimes of glycated protein was measured withexcitation at 370 nm and emission was monitored at 440 nm using timecorrelated single photon counting to be around 0.5, 2.8 and 9.8 ns withvarying relative contributions. Small angle X-ray scattering data showedthe change in inter-particle distances and structural spacing. Dynamicand Static Light Scattering data indicates an increase in particle size,molecular weight and decrease in protein diffusivity. Subsequentmeasurements were at an excitation wavelength of 340 nm and 435 nm.

One biomarker for diabetes related diseases is the formation of AdvancedGlycation Endproducts (AGEs) that result from the Maillard reaction ofproteins with glucose α-crystallin in the ocular lens. Glucoseα-crystallin is a small heat shock protein with no protein turnover and,consequently acts as a record for post-translational modifications,especially glycation, which forms fluorescent AGEs. Steady state andtime resolved fluorescence measurements were used to analyze thespectroscopic changes in alpha crystallin with increase in time ofglycation, in intact lenses from diabetic and non-diabetic donors.Overall, the goal was to develop a non-invasive diagnostic tool forearly detection of diabetes mellitus.

AGEs are formed due to non-enzymatic glycation of the proteins onexposure to open chain sugars and dicarbonyl intermediates.Hyperglycemic conditions, oxidative and thermal stress lead to theformation of Schiff's bases with basic amino acids, for example, lysineand arginine. Further, Amadori products are formed due to rearrangementof the Schiff's bases when highly reactive carbonyl intermediatesaccumulate and attack the amino and guanidine groups on the proteins.Unlike hemoglobin and albumin, the heat shock proteins have longhalf-lives and very low turnover. As a result, the accumulation ofglycation products over a long period of time is of quantitativesignificance.

By screening the changes in the lens proteins and, consequently, theintact lens, non-invasive spectroscopic biomarkers were identified forthe early diagnosis of diabetes mellitus. The appearance of thesebiomarkers will, in turn, act as a standard diagnostic test forcommencing the treatment regimen, thereby preventing furthercomplications.

Establishing a correlation between the structural, molecular andspectroscopic changes in a protein as it slowly denatures due toaggregation induced by non-enzymatic glycation, elucidates theimportance of a glycation model of aging and diabetes and thepathological mechanisms in various degenerative diseases.

Emission spectra were recorded from unmodified and glycated alphacrystallin for the excitation wavelengths set at 340 and 435 nm. Byexcitation at 340 nm, emission spectra showed one distinct peak (FIGS. 1and 6) located at about 440 and 460 nm compatible with the absorptioncaused by AGEs. A broad band was observed at 500 nm by increasing theexcitation wavelength to 435 nm (FIGS. 2 and 37). Fluorescence emissionspectra were also recorded from intact lenses from donors with andwithout diabetics. Qualitatively, the shape and peak value of theemission spectra excited at the same wavelength were very similar forα-crystallin and the intact lenses (FIGS. 3 and 4), indicating thefluorophore formation in lens crystallins.

The fluorescence lifetimes of glycated α-crystallin, when excited at 340and 435 nm can be seen in Tables 1 and 2 respectively. The success ofthe fit was evaluated using reduced chi-square value and Durbin-Watsonparameter. The reduced chi square value was considered good at valuesbetween 0.9-1.2. The Durbin-Watson parameter was 1.6, 1.7 and 1.8 ormore for a one, two and three exponential decay respectively forsuccessful evaluation of the fit. Similarly, the time resolvedfluorescence decay curves from intact lenses at 340 and 435 nm werecollected to obtain the lifetimes of the fluorophores as seen in Tables3, 4, 10-13 respectively.

Time-resolved fluorescence measurements were measured in triplicate andthen the fluorescence decay profiles were analyzed assuming that themodel followed a multiple exponential fit. In the case of 340 nmexcitation, one or two exponential functions did not provide anacceptable fit, as judged by autocorrelation, number of weightedresiduals and Durbin Watson parameters. The lifetimes and their relativecontributions were determined at excitation wavelengths—340 as seen inTable 1. Table 2 shows the fluorescence lifetimes, from unmodified andmodified α-crystallin at an excitation wavelength of 435 nm.

Here, noticeable trends are observed in the lifetimes with glycation ofthe protein. In the visible region, unmodified α-crystallin showed nofluorescence and therefore, no lifetimes. However, for glycatedα-crystallin, new shorter and longer lifetimes appear with increase inthe time of glycation.

For excitation at 340 nm, no change in lifetime was observable withincreasing time of glycation but the amplitude, A1, decreased as theamplitudes, A2 and A3, increased. In addition, the ratio A1/A2 is nearlylinear with time of glycation and serves as a relative measure ofglycation. Table 2 shows the fluorescence lifetimes from unmodified andmodified a-crystallin at an excitation wavelength of 435 nm.

Table 3 provides an overall summary of time resolved fluorescencelifetimes obtained at 340 nm from various donor lenses. Table 4 providesan overall summary of time resolved fluorescence lifetimes obtained byexcitation at 435 nm from various donor lenses. It can be seen that thedistribution of lifetimes and their individual contribution to the totalfluorescence at 435 nm excitation wavelength is very different fornon-diabetic and diabetic donor lenses. While the younger non-diabeticsdid not show any fluorescence, older non-diabetics showed onefluorescence lifetime around 4.7 ns. In the diabetic lenses from youngerdonors, the time resolved fluorescence decay spectra showed a very goodfit with a double exponential decay with lifetimes around 4.6 and 17 ns.The decay spectra from older diabetic donor lenses gave a good fit withtriple exponential decay with lifetimes around 1.4, 4.5 and 16 ns.

Non-enzymatic glycation of α-crystallin is presented as an in vitromodel for aging, diabetes and degenerative diseases.

Alpha crystallin, a small heat shock protein, has been studiedextensively for its chaperone function. α-crystallin sub-units areexpressed in stress conditions and have been found to prevent apoptosisby inhibiting the activation of the caspase pathway. Non-enzymaticglycation of the protein leads to the formation of advanced glycationend-products (AGEs). These AGEs bind to receptors and lead to blockingthe signaling pathways or cause protein precipitation as observed inaggregation related diseases.

Methylglyoxal (MGO) is one of the major glycating agent expressed inpathological conditions due to defective glycolysis pathway. MGO reactsrapidly with proteins, forms AGEs and finally leads to aggregation.Understanding the non-enzymatic glycation induced structural damage inα-crystallin using biophysical and spectroscopic characterization, leadsto develop better disease models for understanding the biochemicalpathways and also in drug discovery.

Non enzymatic glycation of the proteins is a characteristic feature ofaging and diseases like diabetes. This reaction leads to the formationof advanced glycation endproducts on the proteins leading to prolificdamage to their structural integrity. Advanced glycation end-productshave been shown to exhibit very strong fluorescence as the degree ofglycation enhances. The change in fluorescence intensity can serve as aprobe to explain the alterations occurring in the native state proteinand also provides information on the degree of damage done to theprotein.

The augmentation of fluorescence from glycated protein was observed byscreening the fluorescence emission by excitation in UV-A region at 340nm as well as visible region at 435 nm. Oxidative stress, tobaccosmoking and weakened detoxification of AGE precursors have also beenreported as some of the reasons for increase in AGE production. Anotherrisk factor for high AGE production is a diet with high sugar content.Increased AGE levels have also been observed to be major biomarkers inpathophysiology of various degenerative diseases. Compared to sugarslike glucose and sucrose, the dicarbonyl intermediates are much morereactive. The rate of AGE formation depends on the rate of sugarbreakdown into highly reactive intermediates like methyl glyoxal whichin turn depends on many intrinsic and extrinsic factors like defectiveglycolysis pathway, enzymatic peroxidation, deficiency of triosephosphate isomerase, smoking and consumption of high sugar diet.

Steady state fluorescence emission spectra were recorded from intacthuman lenses by excitation at 340 and 435 nm. At 340 nm, the shape andemission maxima were the same in case of non-diabetic lenses but showeda red shift in case of diabetic lenses. However, with increasingexcitation wavelength, the peak emission gradually shifted to longerwavelengths. At 435 nm, fluorescence was observed only in the case oflenses from diabetic donors but not in the non-diabetics. Steady statefluorescence measurements give valuable information about the spectralprofiles. Fluorescence is dependent on various factors likeconcentration of the fluorophores, optical density and instrumentalparameters which are not constant for all the samples. One disadvantageof using fluorescence intensities as a standard is the possibility ofinner filter effect or self-quenching, especially in case of intactlenses where the protein concentration is very high and theconcentration of AGEs increases with age and diabetes.

A better and more reliable alternative is to measure the lifetimes ofAGE fluorophores. The fluorescence lifetime is an intrinsic molecularproperty which is independent of concentration, hence, gives aconsistent, dynamic depiction of the fluorophores. Measuring thefluorescence lifetimes is valuable as the fluorescence decay occurs at ananosecond time scale and is highly influenced by the interactingmolecules as well as their microenvironments. The time resolvedfluorescence decay lifetimes are very specific and also helps analysisof multiple parameters.

At the excitation wavelength of 340 nm, the emission spectra fromglycated α-crystallin has shown a red shift of the emission maxima from420 nm to around 445 nm with increase in extent of glycation. The shiftin the fluorescence emission maxima with time has always been attributedto the formation of new crosslinks within the protein which affects itsconformation and in turn the orientation of the fluorophores in theirmicro-environments. The fluorescence decay observed when the excitationwavelength is 340 nm can be fitted to a sum of three exponentialfunctions.

There are various fluorophores which include photo-degradation productsof tryptophan like N-formyl kynurenine, anthranilic acid, kynurenine,3-hydroxykynurenine, harmane and several advanced glycationend-products. Further, evidence shows that AGEs exhibit photosensitizeractivity in the UV region leading to the rapid oxidation of tryptophan.All of these fluorophores have very similar, overlapping spectralprofiles and hence can be contributing to the increase in fluorescenceintensity with the time of glycation. However, fluorescence lifetimesare highly dependent on the quantum yields and the lifetimes obtainedfrom the fit might be from the tryptophan oxidation products.

With excitation wavelength at 435 nm and an emission maxima between500-510 nm, the influence of tryptophan on the spectral profiles isshown. However, a characteristic difference in the fluorescenceintensity was observed between non-diabetic and diabetic lenses whichfollows the trend observed in unmodified and glycated α-crystallin. Byexciting the diabetic lenses at 435 nm, a significant difference incontribution and also an occurrence of new lifetimes was observed. Theappearance of new lifetimes with increase in age or disease progressionin diabetic lens might be due to the formation of extensive AGEcross-links along with other post-translational modifications.

While, the fundus auto-fluorescence lifetimes are valuable information,these values are dominated by contributions from A2E, FAD and NADH whichare characteristic of age related macular degeneration. Theauto-fluorescence emission from the crystalline lens itself acts as anobstruction for screening the fundus and leads to decrease in fundusautofluorescence (FAF) signal due to back scattering. On the other hand,the fluorescence lifetimes collected from the lens will be primarilyfrom AGE emission without any spectral interference from the connectivetissue making it an effective diagnostic tool for diabetes and otherdiseases associated with AGE formation. The fluorescence lifetimes ofglycated α-crystallin was measured as a protein model and similarconsistent results were observed in the human donor lenses. The exactchemical nature of the dominant fluorophores when excited in the visibleregion is examined Lens auto-fluorescence lifetimes promises to be avery sensitive, noninvasive biomarker for early diagnosis of diabeticeye diseases.

Amplified concentrations of glycating agents in the body causemodifications in glycoproteins as well as heat shock proteins which is acharacteristic feature of aging and diseases like diabetes, cancer andAlzheimer's. The incubation of α-crystallin with different glycatingagents leads to the formation of aggregates over a period of time whichvaries between minutes to years. The aggregates are associated with thedestruction of native state α-crystallin and formation of AGEs which areinitiated by the condensation of basic amino acids and sugars to formSchiff's bases which undergo rearrangement to form Amadori products.

Dynamic Light scattering (DLS) uses visible light to measure the timedependent fluctuations in the scattering intensity to determine thetranslational diffusion coefficient (D_(T)), and hydrodynamic radius(R_(H)) of a particle. The protein solutions have shown very good lightscattering ability and the particle size of native state α-crystallinwas found to be 18±2 nm. The reaction with methyl glyoxal was rapid andthe particle size of the aggregates was around 350 nm within 9 hours(FIG. 5). With an increase in the particle size, the correspondingdiffusivity of the protein decreases. The calculated diffusivity valuesfrom the in vitro study measuring the light scattering from the lens andin turn particle size of the aggregates, predicted the formation of acataract at a very early stage.

Aggregation studies were performed by glycating 1 mg/ml bovineα-crystallin with 1M glucose, 1M sucrose, 0.05 M glycolaldehyde and 10μM methyl glyoxal at 25 oC for different time intervals. The change inparticle size, hydrodynamic radius, diffusion coefficients andrelaxation times of the dilute glycated protein samples were screenedusing DLS with a HeNe laser at 633 nm as in FIGS. 6, 18-22.

Small angle x-ray scattering (SAXS) was used to measure the correlationsbetween alpha-crystallin proteins. SAXS data is very useful indetermining the inter-particle spacing providing insight into thepacking of α-crystallin in very high concentrations withoutsuper-aggregation or crystallization. At low concentrations alphacrystallin shows a monotonic falloff of intensity with scattering vectorq. However, for concentrated suspensions there is a peak that appearsaround q˜0.4 nm⁻¹ for concentrations of 100 mg/ml and moves out toaround q˜0.5 nm⁻¹ at concentrations around 300 mg/ml with increasingconcentration. SAXS was measured from a 250 mg/ml sample of alphacrystallin which showed a strong peak around q=0.42 nm⁻¹ (FIG. 6). Uponglycation with 10 μM methylglyoxal the correlation peak diminishes inintensity and moves to smaller q=0.25 nm⁻¹. SAXS was measured from a 250mg/ml sample of alpha crystallin which showed a strong peak aroundq=0.42 nm⁻¹ FIG. 36. Upon glycation with 10 μM methylglyoxal, thecorrelation peak diminishes in intensity and moves to smaller q=0.25nm⁻¹. This indicates a possible loss of structural integrity of theprotein.

SAXS data is very useful in determining the inter-particle spacing andhence facilitates understanding the packing of α-crystallin in very highconcentrations without super-aggregation or crystallization. Previously,it had been reported that concentrated α-crystallin exhibited a spacingof 14.8 nm by measuring the short range interactions. X-ray diffractionpatterns from SAXS of native α-crystallin led to reports that theprotein exhibits spherical symmetry. Using 250 mg/ml pure α-crystallinhas yielded a single reflection pattern at a Q_(max) of 0.042/Å whichcorresponds to a spacing value of 14.9 nm as shown in FIG. 24. That thepeak maxima, also called iso-scattering point, is the same irrespectiveof the concentration of the protein.

The ANS dye is a valuable tool for the detection of protein surfacehydrophobicity. Usually, the dye has a low fluorescence yield, which isgreatly enhanced on interaction with hydrophobic surfaces. From theexperimental data as shown in FIGS. 7 and 26, a conclusion is thathydrophobicity increases with time of glycation. The spectral shapeexhibits 2 peaks. This can be explained based on the fact that thefluorescence of ANS dye is highly solvent dependent. So the polarity ofthe micro-environment affects the emission maxima of this molecule. ANSdye shows an emission maxima around 550 nm in a highly polar environmentand a blue shift of around 50 nm in a less polar environment. The nativestate α-crystallin, upon glycation unfolds to expose the hydrophobicregions to the surrounding polar environment and due to short rangeinteractions, forms aggregates. Previous literature reports that thechaperone ability of methyl glyoxal modified α-crystallin increasesinitially. However, with increased time of glycation, the proteinaggregates, loses its structural integrity and chaperone ability. As aresult, other lens proteins are easily prone to stress leading to theirdamage. Structural studies disclosed here help explain the loss ofchaperone activity, thereby establishing the relation between structureand function of alpha crystallin.

Progressive loss of soluble α-crystallin, associated with increasedhydrophobicity and formation of aggregates is responsible for increasingthe lens stiffness. Heat induced denaturation of α-crystallin may be animportant factor in the etiology of presbyopia. However, considering thephysiological conditions, non-enzymatic glycation seems to be the mainculprit. Cataract and presbyopia can produce myopia due to a change inthe refractive index of the crystalline lens of the eye. The presence ofhigh molecular weight aggregates and also advanced glycationend-products lead to a change in both the scattering intensities as wellas the spectroscopic properties of the lens. Due to change in refractiveindex of the lens, the angle of the refracted light changes leading toseveral focal points in close proximity on the retina causing chromaticaberrations.

Change in solubility parameters of the lens proteins with increase intime of glycation have been studied by screening the change in water andurea soluble portions using UV-Vis spectrophotometry at 280 nm. FIG. 27shows that the glycation was associated with significant and progressiveinsolubility of the lens proteins over the period of time.

Cataract and presbyopia can produce myopia due to a change in therefractive index of the crystalline lens of the eye. The refractiveindex of native α-crystallin which is close to that of water alsochanges as shown in FIG. 28. The presence of high molecular weightaggregates and also advanced glycation end-products leads to a change inboth the scattering intensities as well as the spectroscopic propertiesof the lens. Due to change in refractive index of the lens, the angle ofthe refracted light changes leading to several focal points in closeproximity on the retina causing chromatic aberrations.

Tryptophan has a very strong fluorescence in proteins. The residueswhich are buried in the hydrophobic core of proteins can have spectrawhich are shifted by 10 to 20 nm compared to tryptophans on the surfaceof the protein. Tryptophan fluorescence can be quenched due tomicroenvironments. The magnitude of fluorescence intensity is a probe toexplain the perturbations occurring in the native state. The wavelengthmaxima of tryptophan fluorescence shifted on modification indicating achange in the microenvironment which was confirmed by surfacehydrophobicity measurements (FIG. 7). In α-crystallin, tryptophans arenot located at the N-terminus and hence do not have a free amino groupto participate in the Maillard reaction. The photo degradation productsof alpha crystallin showed that they were primarily from tryptophanoxidation.

Another aspect of tryptophan fluorescence spectral profiles is a shiftin the isosbestic point of the spectra. Usually, the presence of anisosbestic point indicates that only two species that vary inconcentration contribute to the absorption around the isosbestic point.If a third molecule is participating in the process, the spectratypically intersect at varying wavelengths as concentrations change,creating the impression that the isosbestic point is “out of focus”, orthat it will shift as conditions change. The reason for this is that thedifferent compounds have varying extinction coefficients at oneparticular wavelength. Tryptophan oxidation products have spectralprofiles similar to AGEs with varying extinction coefficients. Thishelps explain the decrease in tryptophan fluorescence with increase inthe time of glycation.

The rate of AGE formation depends on the rate of the formation of highlyreactive intermediates due to defective glycolysis pathway andconsumption of high sugar diet. The change in AGE fluorescence withdecrease in tryptophan fluorescence can be observed in FIG. 8.

Tryptophan (Trp) lifetime measurements can be used to provideinformation about the changes in the microenvironments. Tryptophanfluorescence is a very valuable tool that can be applied to studyfolding/unfolding, the effect of environment and solvent exposure on theintegrity of the protein. The fluorescence lifetimes of tryptophan canbe used to analyze the location of tryptophan and the possibility ofshort range interactions. The inventors have shown that the fluorescenceof α-crystallin is predominantly due to the Trp 9 in both A and B chainswhile the Trp 60 present in the B chain is buried from exposure.However, after the process of glycation begins, the tryptophans appearto become completely exposed to the surrounding aqueous medium. This canbe confirmed from the lifetime measurements as in Table 2, no changewere observed in the lifetimes with time of glycation. The tryptophanresidues may undergo a shift in their position after once the proteinstarts to unfold and aggregate leading to the formation of waterinsoluble residues.

The Trp residues in peptides generally show fluorescence emissionspectra, with a peak around the 340-370 nm region, similar to the Trpresidues in aqueous media or in fully denatured proteins. However,interaction of peptides possessing Trp residues with proteins wouldresult in diminished exposure of the peptide Trp (as seen in Trp 60) tothe aqueous environment with a concomitant blue shift in thefluorescence emission maxima. Simulation studies have shown that,although Trp 60 is exposed to solvent in the α-B subunit, it is buriedin the dimer models, located at the interacting interface of thesubunits. Using LCMS and isotopic labeling, reports have shown that K70,K88, K92, K99, K103, K145, K150 and K166 are the lysine glycationtargets on α-crystallin. R12, R21, R54, R65, R69, R103, R112, R117,R119, R157 and R163 are arginine glycation sites on α-crystallin. Basedon these target sites of glycation, modifications may occur within thevicinity of the tryptophan (Trp 60) exposing it to differentmicroenvironments as they are chemically modified.

Approximate molecular masses of modified and unmodified bovineα-crystallin samples were determined using Static Light Scattering(SLS). A Zimm plot was constructed from the light scattering intensitiesobtained at multiple angles (10, 20, 30, 40, 50, 60, 70, 80, 95, 100,110 and 120) and different concentrations (0.125, 0.25, 0.5 and 1 mg/ml)to obtain the approximate molecular weights (FIG. 23). Small angle X-rayscattering (SAXS) was used to measure the inter-particle distances asX-rays due to their shorter wavelength can interact with atoms (FIG. 24,25) and also facilitate understanding the shape of the molecules. Theselight scattering techniques reveal physical changes in the protein andthe effect of modifications on the structure and shape of the protein.

Secondary structure is determined by circular dichroism spectroscopy inthe “far-UV” spectral region (190-250 nm). At these wavelengths thechromophore is the peptide bond, and the signal arises when it islocated in a regular, folded environment. α-crystallin is mainly made ofbeta sheets and hence the signal is observed as a downward trough ataround 220 nm. With increase in period of glycation, unfolding occursleading to formation of random coil (FIG. 38).

Tryptophan

One of the major fluorophores in proteins is tryptophan. With increasein glycation, tryptophan fluorescence decreases (FIG. 30). But,tryptophans in α-crystallin do not undergo chemical modification due toMaillard reaction as there are no free amino groups. Tryptophanfluorescence is a very valuable tool that can be applied to studyfolding/unfolding, the effect of environment and solvent exposure on theintegrity of the protein. The fluorescence lifetimes of tryptophan canbe used to analyze the location of tryptophan and the possibility ofshort range interactions (Table 5).

FIGS. 10, 11, 12, 33 provide case studies comparing the spectroscopichotspots in lens autofluorescence for similar aged donor non-diabeticand diabetic lenses. Emission spectra were recorded for the excitationwavelengths set at 340, 370 and 435 nm. By excitation at 340 and 370 nm,emission spectra showed one distinct peak (FIGS. 34 and 35) located atabout 440 and 460 nm compatible with the absorption caused by AGEs. Abroad band was observed at 500 nm by increasing the excitationwavelength to 435 nm (FIG. 36). Fluorescence emission spectra were alsorecorded from intact lenses from donors with and without diabetics.Qualitatively, the shape and peak value of the emission spectra excitedat the same wavelength were very similar for α-crystallin and the intactlenses (FIGS. 4 and 5), indicating the fluorophore formation in lenscrystallins.

Time-resolved fluorescence measurements were analyzed from thefluorescence decay assuming that the model followed a triple exponentialfit. In the case of 340 and 370 nm excitation, one or two exponentialfunctions did not provide an acceptable fit, as judged byautocorrelation, number of weighted residuals and Durbin Watsonparameters. The lifetimes and their relative contributions weredetermined at excitation wavelengths—340 and 370 nm as seen in Tables 8and 9. The overall fluorescence lifetimes were longer for excitation at370 nm but they did not change with time of glycation in either cases.Table 2 shows the fluorescence lifetimes from unmodified and modifiedα-crystallin at an excitation wavelength of 435 nm. There are noticeabletrends in the lifetimes with glycation of the protein. In the visibleregion, unmodified α-crystallin showed no fluorescence and therefore, nolifetimes. However, for glycated α-crystallin, new shorter and longerlifetimes were observed with increase in the time of glycation.

Steady state and time resolved fluorescence measurements were collectedfrom intact non-diabetic and diabetic lenses ranging from 6 to 92 yearold donors. An illustration of difference in the spectral profiles fromdonor lenses of 3 different age groups can be seen in FIGS. 4 and 5.Table 3 provides an overall summary of time resolved fluorescencelifetimes obtained at 340 nm from various donor lenses. A sample of timeresolved fluorescence data comparisons from non-diabetic and diabeticdonor lenses from different age groups can be observed in Tables 10-13.Table 4 provides an overall summary of time resolved fluorescencelifetimes obtained by excitation at 435 nm from various donor lenses.From this data, it can be seen that the distribution of lifetimes andtheir individual contribution to the total fluorescence at 435 nmexcitation wavelength is very different for non-diabetic and diabeticdonor lenses. Although the younger non-diabetics did not show anyfluorescence, older non-diabetics showed one fluorescence lifetimearound 4.7 ns. In the diabetic lenses from younger donors, the timeresolved fluorescence decay spectra showed a very good fit with a doubleexponential decay with lifetimes around 4.6 and 17 ns. The decay spectrafrom older diabetic donor lenses gave a good fit with triple exponentialdecay with lifetimes around 1.4, 4.5 and 16 ns.

Amplified concentrations of glycating agents in the body causemodifications in glycoproteins as well as heat shock proteins which is acharacteristic feature of aging and diseases like diabetes, cancer andAlzheimer's. AGE formation was evaluated in vitro through incubation ofα-crystallin with various glycating agents, for example glucose,sucrose, glycolaldehyde (GA) and methyl glyoxal (MGO). Tryptophan andAGE fluorescence was used to evaluate α-crystallin damage. \

The protein solutions have shown very good light scattering ability andthe particle size of native state α-crystallin was found to be 18±2 nm.When incubated with glucose and sucrose, there was no effect on theparticle size of α-crystallin over a period of 5 weeks. Withglycolaldehyde, the particle size slowly increased from 18 to 350 nmover a period of 5 weeks when incubated at 25° C. The reaction with MGOwas relatively faster and the particle size of the aggregates was around350 nm within 9 hours (FIG. 6). The change in the absorbance andappearance of new excitation maxima with increase in time of glycationis shown in FIGS. 18 and 19 for GA and MGO respectively. As there was anincrease in the particle size, the corresponding diffusivity of theprotein decreased. By measuring the light scattering from the lens andin turn particle size of the aggregates, the formation of a cataract ispredicted at a very early stage. The calculated diffusivity values fromthe in vitro study agree with the results obtained from the in vitrostudy to determine the protein diffusivity in rabbit lenses. Bymeasuring the light scattering from the lens and in turn particle sizeof the aggregates, the formation of a cataract at a very early stage canbe predicted.

A large increase in the molecular weight of α-crystallin was followedusing static light scattering (FIG. 25). A cataract may be amanifestation of high molecular weight protein aggregates. Aggregatesmay be formed by lens protein cross links linked by disulfide orcovalent bonds as monitored by gel electrophoresis and massspectrometry. These aggregates may be present randomly in the lens andhave an ability to diffuse freely leading to increase of lightscattering and thereby change in the refractive index of the lens.

Increase in temperature is associated with thermal aggregation as wellas increase in the spacing. The effect of glycation on short rangeinteractions and spacing, X-ray scattering profiles of 250 mg/mlα-crystallin glycated with 10 μM methyl glyoxal were studied as shown inFIG. 27. A shift and formation of a weak peak was observed at lowerscattering angles with a Q_(max) of 0.025/Å which gives a spacing valueof 25.1 nm. The weak scattering peak maxima is likely due to loss ofstructural integrity of the protein and spherical symmetry of thehetero-oligomeric complex.

Molecular chaperones are a class of proteins which help in the refoldingof unfolded proteins to their native state. In the absence ofchaperones, these unfolded proteins may mutually associate via exposedhydrophobic regions and precipitate out of solution. Chaperones have animportant role in protein folding and refolding by stabilizing theunfolded proteins, hence protecting them from stress conditions.α-crystallin has been shown to function as a molecular chaperone inpreventing thermal aggregation of crystallins and other proteins. ANSfluorescence for surface hydrophobicity has been used for thespectroscopic investigation of the high molecular weight complex formedas a result of glycating bovine α-crystallin The surface hydrophobicityincreases with the time of glycation providing more hydrophobicsurfaces. The chaperone ability of methyl glyoxal modified α-crystallinincreases initially. However, with increased time of glycation, theprotein aggregates, loses its structural integrity and chaperoneability. As a result, other lens proteins are easily prone to stressleading to their damage. Structural results disclosed herein explain theloss of chaperone activity reported by others, thereby establishing therelation between structure and function of alpha crystallin.

The formation of aggregates, change in inter-particle spacing anddecreased protein diffusivity also affect the physicochemical parametersof the proteins. The intricate complexation and arrangement ofα-crystallin is responsible for the transparency of the lens. FIG. 29shows that the soluble portion of the protein decreases drastically overtime. At the same time, the refractive index of native α-crystallinwhich has to be close to that of water also changes as shown in FIG. 30.Change in refractive index leads to increased light scattering in theeye and may be the reason for chromatic aberration that occurs withcataracts and in presbyopia. Overall, the formation of high molecularweight aggregates due to glycation leads to a change in both thescattering intensities as well as the spectroscopic properties of thelens.

Far UV circular dichroism (CD) spectra reflect the changes in secondarystructure of the proteins. Both the native state and glycatedα-crystallin showed a minimum at 217 nm which indicates beta sheets(FIG. 32). However, with the progression of glycation, α-crystallinshowed a considerable decrease in the negative ellipticity and atendency to shift towards the left. Loss of structural stability due tothe formation of random coils can be concluded from the CD data. Thiscan be explained by the formation of high molecular weight aggregatesand increase in surface hydrophobicity. Presence of these residues inthe random coil may explain the increase in hydrophobicity of theprotein associated with the loss of beta sheets.

Tryptophan has a very strong fluorescence in the proteins. The residueswhich are buried in the hydrophobic core of proteins can have spectrawhich are shifted by 10 to 20 nm compared to tryptophans on the surfaceof the protein. Tryptophan fluorescence is quenched due tomicroenvironments. In case of α-crystallin, the unmodified protein seemsto have more fluorescence than the modified as shown in FIG. 32. Themagnitude of fluorescence intensity can serve as a probe to explain theperturbations occurring in the native state. The wavelength maxima oftryptophan fluorescence shifted on modification indicating a change inthe microenvironment which can be confirmed by surface hydrophobicitymeasurements (FIG. 28). In α-crystallin, tryptophans are not located atthe N-terminus and hence do not have a free amino group to participatein the Maillard reaction. The photo degradation products of alphacrystallin are primarily from tryptophan oxidation.

There is a shift in the isosbestic point of the spectra. Usually, thepresence of an isosbestic point indicates that only two species thatvary in concentration contribute to the absorption around the isosbesticpoint. If a third molecule is participating in the process, the spectratypically intersect at varying wavelengths as concentrations change,creating the impression that the isosbestic point is ‘out of focus’, orthat it will shift as conditions change. The reason for this is that thedifferent compounds have varying extinction coefficients at oneparticular wavelength. Tryptophan oxidation products have spectralprofiles similar to AGEs with varying extinction coefficients.Tryptophan fluorescence decreased with increase in the time ofglycation.

Advanced glycation end-products have a characteristic fluorescence andare excited in the spectral region of tryptophan emission. Generation ofAGEs can be associated with oxidative stress, tobacco smoking andweakened detoxification of AGE precursors. Compared to sugars likeglucose and sucrose, the dicarbonyl intermediates cause a lot of damage.This indicates that the rate of AGE formation also depends on the rateof breakdown of sugars into the highly reactive intermediates which inturn is dependent on many intrinsic and extrinsic factors. The change inAGE fluorescence with decrease in tryptophan fluorescence can beobserved in FIG. 30 and FIG. 32.

Tryptophan (Trp) lifetime measurements is used to give a rough ideaabout the changes in the microenvironments. Tryptophan fluorescence is avery valuable tool that can be applied to study folding/unfolding, theeffect of environment and solvent exposure on the integrity of theprotein. The fluorescence lifetimes of tryptophan can be used to analyzethe location of tryptophan and the possibility of short rangeinteractions. Fluorescence of α-crystallin is predominantly due to theTrp 9 in both A and B chains while the Trp 60 present in the B chain isburied from exposure. However, after the process of glycation begins,the tryptophans are completely exposed to the surrounding aqueousmedium. This can be confirmed from the lifetime measurements as in Table5, no change was observed in the lifetimes with time of glycation.However, the measurements hint at a possibility that the tryptophanresidues undergo a shift in their position once the protein starts tounfold and aggregate leading to the formation of water insolubleresidues.

Predicted flow velocity is extremely slow considering the nutrientstransported increase the viscosity of the fluid. This is in tune withthe very low diffusion coefficient data obtained from dynamic lightscattering data. Glucose and sucrose pass through the lens but do notspontaneously react with the proteins. On the other hand, glycolyticintermediates like methyl glyoxal are extremely reactive and startforming Schiff's bases when they come in contact with the protein andinstantaneously lead to the formation of AGEs. Also, the aggregatescause an irreparable damage to the intricate protein network causingopacification which is most commonly observed in case of cataracts.

Non enzymatic glycation of the proteins is a characteristic feature ofaging and diseases like diabetes.

The main contributing fluorophore when excited in the UV region isreported to be argpyrimidine. The effect of different microenvironmentson the emission spectrum and fluorescence lifetimes explains the redshift. However, the fluorescence lifetimes did not change with time ofglycation. This shows that the fluorescence lifetimes measured in thisregion are not affected by glycation.

Materials and Methods

Methyl glyoxal was purchased from Sigma Aldrich. Fresh bovine lenseswere obtained from the Aurora meat packing company, Illinois. Wholehuman donor lenses were obtained from the Eye Bank within 2 days afterdeath. The lenses from donor age range of 40-90 years were used for thisstudy. The lenses were stored at −70° C. in the dark till the day ofuse.

A) Extraction of Bovine Alpha Crystalline:

Fresh bovine lenses were weighed and then homogenized by stirring in abuffer prepared using 50 mM Tris/0.2 M NaCl/1 mM EDTA/10 mMmercaptoethanol, pH 7.4 at 4° C. The supernatant was centrifuged at14000 g for 1 hr. at 4° C. Alpha-crystallin was isolated from the totalsoluble lens protein solution by size exclusion chromatography. A totalof 30 ml supernatant was loaded on a 1.7 cm×100 cm CL-6B sepharose gelfiltration column, and using a peristaltic pump, eluted at a flow rateof 1 ml/min and monitored by absorbance at 280 nm. Alpha crystallinelutes first as a single symmetrical peak at approximately 170 ml of thebuffer corresponding to an apparent molecular mass of 800 KDa. Theisolated a crystallin fractions were pooled and desalted withUltrafree-15 Biomax-10K centrifugal filter devices (MilliporeCorporation, Bedford, Mass.) at a speed of 3000 rpm by rinsing threetimes with water. The purity of the sample was determined by SDS-PAGEwith the Pharmacia LKB*Phast System and by mass spectrometry (45).

B) Sample Preparation:

Bovine alpha crystallin was used as the model protein to study theprogression of glycation. Methyl glyoxal (MGO) was used as the glycatingagent at a concentration of 2.5 μM in 1 mg/ml alpha crystallin solutionand incubated at 25° C. over a period of 60 days. The aliquots werecollected at regular intervals and dialyzed against 10 mM phosphatebuffer, pH 7.4.

C) Steady State Fluorescence Measurements:

The AGE fluorescence of unmodified and methyl glyoxal modifiedalpha-crystallin was measured using Hitachi F2500 FluorescenceSpectrometer. The fluorescence emission spectrum of the samples wasrecorded using 340 and 435 nm as excitation wavelength maintaining theslit widths at 5 nm and PMT voltage of 400V. The same procedure wasrepeated using the whole donor lenses to study the spectral propertiesof the fluorophores.

D) Time Resolved Fluorescence Measurements:

The lifetime profiles for the fluorescence decay of fluorophores inunmodified and modified alpha crystallin (10 mM phosphate buffer at pH7.4) were measured in triplicate by using TimeMaster™ LED system(TM-2000). The samples were excited in UV-A region at 340 nm and invisible region at 435 nm using a LED light source having approximately1.5 ns pulse width. The time domain system was used to measure the decayin fluorescence with respect to time. The raw decay data was analyzedusing global 1 to 4 three exponential fitting analysis withdeconvolution which gives the lifetimes as well as the relativecontributions to the total fluorescence at time zero. These data wereassessed with a good auto correlation function around zero, weightednumber of residuals randomly distributed between +3 and −3, reduced

² values between 0.9 to 1.1 and the Durbin-Watson parameters of greaterthan or equal to 1.6, 1.7 and 1.8 for one, two and three exponentialdecay respectively. Once the lifetimes were obtained using the modelprotein, the procedure was extended to measure the fluorescencelifetimes of the intact lenses of diabetic and non-diabetic donors. Thedonor lenses were positioned parallel to excitation source and theemission was detected at right angles to the excitation beam.

Methyl glyoxal was purchased from Sigma Aldrich. Fresh calf lenses wereobtained from the Brown Packing Co. (South Holland, Ill.).

E) Extraction of Bovine Alpha Crystallin:

Fresh calf lenses were weighed and then homogenized by stirring in abuffer prepared using 50 mM Tris/0.2 M NaCl/1 mM EDTA/10 mMmercaptoethanol, pH 7.4 at 4° C. The supernatant was centrifuged at14000 g for 1 hr at 4° C. α-crystallin was isolated from the totalsoluble lens protein solution by size exclusion chromatography. A totalof 30 ml supernatant was loaded on a 1.7 cm×100 cm CL-6B sepharose gelfiltration column, and using a peristaltic pump, eluted at a flow rateof 1 ml/min and monitored by absorbance at 280 nm. Alpha crystallinelutes first as a single symmetrical peak at approximately 170 ml of thebuffer corresponding to an apparent molecular mass of 800 KDa. Theisolated a crystallin fractions were pooled and desalted withUltrafree-15 Biomax-10K centrifugal filter devices (MilliporeCorporation, Bedford, Mass.) at a speed of 3000 rpm by rinsing threetimes with water. The purity of the sample was determined by SDS-PAGEwith the Pharmacia LKB*Phast System and by mass spectrometry.

F) Sample Preparation:

Bovine α-crystallin was used as the model protein to study theprogression of glycation. Methyl glyoxal (MGO) was used as the glycatingagent at a concentration of 10 μM in 1 mg/ml alpha crystallin solutionand incubated at 37° C. over a period of 9 hours. The aliquots werecollected at 0.5, 1, 3, 6, 9 hr. The samples were dialyzed against 10 mMphosphate buffer, pH 7.4, lyophilized and reconstituted with 1 ml of MQwater before measurements.

G) Dynamic Light Scattering Measurements (DLS)

Particle sizes, and particle cluster sizes were measured using DLS on aBrookhaven BI-200SM Research Goniometer and Laser Light ScatteringSystem. For a spherical particle diffusing through a solution ofviscosity η the intensity correlation function (g₂) measured through DLSdecays with an exponential relaxation rate, g₂(t)=1+β exp(−2Γt). Theparticle size can be obtained from the decay rate via R=k_(B)Tq²/6πηΓ.Here k_(B) is Boltzmann's constant, T is the temperature and

$q = {4\pi \; {{\sin \left( \frac{\theta}{2} \right)}/\lambda}}$

is the scattering vector of the laser light scattered at θ=90° and λ=632nm.

For a suspension consisting of a mixture of particles of varying sizethe distribution of sizes can be obtained from an inverse Laplacetransform. We used the CONTIN software package to obtain particle sizedistributions in this manner. The accuracy of the distributions wasdetermined based on the polydispersity and the baseline difference fromthe correlation curve.

H) Surface Hydrophobicity Measurements

The surface hydrophobicity of the native and MGO modified alphacrystallin was studied using a specific hydrophobic probe,1-anilinonaphthalene-8-sulfonic acid (ANS). Ten microliters of a 10 mMmethanolic solution of ANS was added to 1 mL of protein [0.1 mg/mL in 10mM phosphate buffer (pH 7.4)], and the mixture was incubated for 1 hr inthe dark at 25° C. Fluorescence emission spectra were recorded between400 and 600 nm using an excitation wavelength of 370 nm. The excitationand emission band-passes were 5 nm each.

I) Small Angle X-Ray Scattering Measurements

Concentrated samples of alpha crystallin and glycated alpha crystallinhave been studied using SAXS. These studies were performed at the 8-ID-Ibeam line of the Advanced Photon Source at Argonne National labs, IL.The sample in a sealed capillary was placed in the beam line undervacuum. Coherent X ray photons of energy 7.35 keV were focused using aKinoform lens placed upstream of the sample. The scattered photons weredetected by a PI LCX-1300 direct detection CCD (Princeton Instruments,USA) 4.0 m downstream of the sample. A single camera width spans about0.3 nm⁻¹ in the scattering vector (Q) space. The intensities weremeasured at 6 overlapping camera widths covering a Q range from 0.1-1nm⁻¹. For a given camera position, 20 frames were collected with anexposure time of 0.2 s for each frame. The shorter exposures of 0.2 sand moving the beam spot to a different location and camera to a newposition helps to minimize the radiation damage caused by the X-raybeam. The resulting image pixels were analyzed by a MATLAB GUI to obtainthe time averaged intensity profiles as a function of the scatteringvector. The inter-particle distance between the sub-units can bedetermined from scattering vector Q using Q_(max)=2π/d; where,d=distance between adjacent sub-units.

J) Steady State Fluorescence Measurements:

The tryptophan fluorescence of unmodified and methyl glyoxal modifiedα-crystallin was measured using Hitachi F2500 Fluorescence Spectrometer.The fluorescence emission spectrum of the samples was recorded between300-700 nm using 295 nm as excitation wavelengths maintaining the slitwidths at 2.5 nm and PMT voltage of 400V.

K) Time Resolved Fluorescence Measurements

The lifetime profiles for the tryptophan fluorescence decay inunmodified and modified alpha crystallin (10 mM phosphate buffer at pH7.4) were measured by using TimeMaster™ LED system (TM-2000). Thesamples were excited at 295 nm using a LED light source havingapproximately 1.5 ns pulse width. The time domain system was used tomeasure the decay in fluorescence with respect to time. The raw decaydata was analyzed using global 1 to 4 three exponential fitting analysiswith deconvolution which gives the lifetimes as well as the relativecontributions to the total fluorescence at time zero. These data wereassessed with a good auto correlation function around zero, weightednumber of residuals randomly distributed between +3 and −3, reduced

² values around 0.9 to 1.1.

TABLE 1 Time resolved fluorescence lifetimes from glycated α-crystallinby excitation at 340 nm and emission maxima between 420-440 nm. Time ofglycation Durbin - with MGO Lifetimes,

 (ns) and relative contributions at time zero, A (%)

Watson (days) A₁

A₂

A₃

value parameter Control 70.5 ± 1.5 0.5 ± 0.04 18.9 ± 1.9  2.4 ± 0.0210.6 ± 3.2  7.2 ± 0.17 0.96 1.83 10 65.5 ± 1.4 0.6 ± 0.15 22.1 ± 1.2 2.2± 0.3 12.4 ± 0.8 7.2 ± 0.5 1.04 1.87 20 54.8 ± 2.9 0.6 ± 0.01 29.5 ± 1.7 2.5 ± 0.07 15.7 ± 1.2  7.6 ± 0.02 0.98 1.8 30 49.8 ± 2.9 0.7 ± 0.08  34 ± 2.7 2.8 ± 0.3 16.2 ± 2.8  7.9 ± 0.32 1.031 1.92 40 45.3 ± 0.1 0.5± 0.1   37.7 ± 2.1 2.3 ± 0.2   17 ± 0.95 7.9 ± 0.1 1.002 1.94 50 34.9 ±1.1 0.7 ± 0.1   45.2 ± 1.2 2.4 ± 0.3 19.9 ± 0.7 7.85 ± 0.5  0.954 1.8160 28.8 ± 4.5 0.76 ±     50.1 ± 3.2 2.78 ± 0.4  21.1 ± 1.7   8 ± 0.60.97 2.01

TABLE 2 Time resolved fluorescence lifetimes from glycated α-crystallinby excitation at 435 nm and emission maxima between 500-510 nm. Time ofglycation Durbin - with MGO Lifetimes, 

 (ns) and relative contributions at time zero, A (%)

Watson (days) A₁

A₂

A₃

value parameter Control — — — — — — — — 10 — — 100 4.7 ± 0.04 — — 0.9991.635 20 — — 96.11 ± 1.6  4.76 ± 0.2  3.89 ± 0.06 19.5 ± 1.7 0.985 1.7530 44.1 ± 0.3 1.67 ± 0.1  20.1 ± 0.2  4.51 ± 0.09  35.8 ± 0.6 17.6 ± 0.51.07 1.81 40 45.1 ± 0.2 1.6 ± 0.11 19.7 ± 0.31 4.6 ± 0.04 35.2 ± 0.01  17 ± 0.6 1.075 1.871 50 58.2 ± 0.2 1.4 ± 0.43 30.1 ± 0.3  4.6 ± 0.1411.8 ± 2.7 16.7 ± 0.3 1.03 1.85 60 51.6 ± 0.2 1.5 ± 0.31 42.3 ± 0.64 4.4± 0.43 6.07 ± 0.3 15.6 ± 1.4 0.987 1.961

TABLE 3 Summary for time resolved fluorescence lifetimes from humandonor lenses by excitation at 340 nm and emission maxima between 420-440nm. Durbin - Types of Lifetimes, 

 (ns) and relative contributions at time zero, A (%)

Watson donor lens A₁

A₂

A₃

value parameter Non-diabetic 52-65% 0.62 ± 0.1 20-33% 2.6 ± 0.14 13-21%7.6 ± 0.2 1.04 1.97 (n = 35) Diabetic 38-49% 0.66 ± 0.1 37-45% 2.3 ±0.1  12-19%  7.6 ± 0.12 1.01 1.9 (n = 9)

TABLE 4 Summary for time resolved fluorescence lifetimes from humandonor lenses by excitation at 435 nm and emission maxima between 500-510nm. Durbin - Types of Lifetimes, 

 (ns) and relative contributions at time zero, A (%)

Watson donor lens A₁

A₂

A₃

value parameter Nondiabetic* — — — — — — — — (<45 year) (n = 10)Nondiabetic — — 100 4.76 ± 0.02  — — 0.967 2.14 (>45 year) (n = 34)Nondiabetic — — 58.1 ± 0.5 4.6 ± 0.14 41.9 ± 0.2 16.2 ± 0.05 0.992 1.76(>45 year) (n = 4) Diabetic 37.1 ± 4.8 1.6 ± 0.07 33.9 ± 5.2 4.3 ± 0.02  29 ± 2.6 15.1 ± 0.01 0.985 2.06 (>45 year) (n = 5) *No diseaseconditions and ocular history.

TABLE 5 Changes in tryptophan lifetimes of α-crystallin after incubatingwith using 10 μM methylglyoxal over a period of 9 h at 25° C. Lifetimes,τ (ns) and relative Time of glycation with contributions at time zero, A(%)

methyl glyoxal (h) A₁ τ₁ A₂ τ₂ A₃ τ₃ value 0 16.1 1.3 53.4 4.3 30.5 9.60.97 0.5 12.3 1.5 48.5 4 39.2 9.8 1.11 1 14.7 1.5 44.4 5 40.9 9.8 1.08 316.1 1.7 42.3 4.9 39.5 9.8 1.21 6 17.4 1.8 48.2 4.9 34.4 10 1.01 9 18.22.3 43.7 3.5 40.2 10 0.99

TABLE 6 Sequences MDIAIHHPWIRRPFFPFHSPSRLFDQFFGEHLLESDLFPTSTSLSPFYLRPPSFLRAPSWFDTGLS[EMRLEK DRFSVNLDVKHFSPEELKVK VLGDVIEVHGKHEERQDEHGFISREFHRKYRIPADVDPLTITSSLSSDGVLTVNGPRKQVSGPERTIPI]TREEKPAVTAAPKK Black bold—αA- crystallin recognitionsites on αB- crystallin Underline—chaperone site Bracketed—α -crystallindomain (Sreelakshmi, Santhoshkumar, Bhattacharyya, & Sharma, 2004)

TABLE 7 Criteria for the diagnosis of diabetes and of increased risk fordiabetes [pre-diabetes]/Impaired Fasting Glucose [IFG] adopted from ADA-Clinical Practice Recommendations [Diabetes Care, 36 [S1], 2013 ADA Goalfor Diagnosis of Increased risk Diabetes Test* Diabetes[Prediabetes]/IFG HbA1c ≥6.5% 5.7-6.4% Using a method certified by NGSPand standardized to the DCCT assay. or Fasting Plasma Glucose ≥126 mg/dL100-125 mg/dL Fasting is defined as no (7.0 mmol/L) (6.9 mmol/L) caloricintake for at least 8 hours. or 2 Hour Plasma Glucose ≥200 mg/dL 140-199mg/dL [OGTT] The test should be (11.1 mmol/L) (7.8-11.0 mmol/L)performed as described by the WHO, using a glucose load containing theequivalent of 75 g anhydrous glucose dissolved in water.

TABLE 8 Time resolved fluorescence lifetimes from glycated alphacrystallin by excitation at 340 nm and emission maxima between 420-440nm Time of glycation with Lifetimes, 

 (ns) and Durbin - MGO Relative contributions at time zero, A (%) Watson(days) A₁

A₂

A₃

 value parameter Control 70.5 ± 1.5 0.5 ± 0.04 18.9 ± 1.9  2.4 ± 0.0210.6 ± 3.2 7.2 ± 0.17 0.96 1.83 10 65.5 ± 1.4 0.6 ± 0.15 22.1 ± 1.2 2.2± 0.3 12.4 ± 0.8 7.2 ± 0.5  1.04 1.87 20 54.8 ± 2.9 0.6 ± 0.01 29.5 ±1.7  2.5 ± 0.07 15.7 ± 1.2 7.6 ± 0.02 0.98 1.8 30 49.8 ± 2.9 0.7 ± 0.08  34 ± 2.7 2.8 ± 0.3 16.2 ± 2.8 7.9 ± 0.32 1.031 1.92 40 45.3 ± 0.1 0.5± 0.1  37.7 ± 2.1 2.3 ± 0.2   17 ± 0.95 7.9 ± 0.1  1.002 1.94 50 34.9 ±1.1 0.7 ± 0.1  45.2 ± 1.2 2.4 ± 0.3 19.9 ± 0.7 7.85 ± 0.5  0.954 1.81 6028.8 ± 4.5 0.76 ± 0.2  50.1 ± 3.2 2.78 ± 0.4  21.1 ± 1.7  8 ± 0.6 0.972.01

TABLE 9 Time resolved fluorescence lifetimes from glycated alphacrystallin by excitation at 370 nm and emission maxima between 440-460nm. Time of glycation with Lifetimes, 

 (ns) and Durbin - MGO Relative contributions at time zero, A (%) Watson(days) A₁

A₂

A₃

 value parameter Control 40.5 ± 1.8 1.1 ± 0.1 39.2 ± 0.1 3.6 ± 0.17 20.3± 2.6 10.1 ± 1.7 0.97 1.82 10 41.9 ± 1.4 1.2 ± 0.2 37.9 ± 0.01 3.7 ±0.14 20.2 ± 0.6 10.6 ± 0.7 0.997 1.945 20 42.8 ± 2.9 1.3 ± 0.31 36.1 ±1.6 3.67 ± 0.1  21.1 ± 0.7 10.5 ± 1.7 0.99 1.85 30 44.1 ± 0.3 1.16 ± 0.234.1 ± 0.2 3.55 ± 0.09  21.8 ± 0.6 10.6 ± 0.5 1.01 1.801 40 45.1 ± 0.21.2 ± 0.11 31.7 ± 0.31 3.6 ± 0.24 23.2 ± 0.01 10 ± 0.6 0.985 1.917 5049.2 ± 0.2 1.0 ± 0.43 29.1 ± 0.3 3.6 ± 0.19 21.7 ± 2.7 10.7 ± 0.3 1.0131.95 60 51.6 ± 0.2 1.1 ± 0.39 27.3 ± 0.64 3.4 ± 0.41 21.1 ± 0.3 10.6 ±1.4 0.988 1.9

TABLE 10 Comparison of time resolved fluorescence lifetimes from 20 yrold non-diabetic and 18 yr old type I diabetic human donor lenses byexcitation at 435 nm and emission maxima between 500-510 nm TypeLifetimes, 

 (ns) and Durbin - of donor Relative contributions at time zero, A (%)Watson lens A₁

A₂

A₃

 value parameter Non- — — — — — — — — diabetic (20 yr) Diabetic — — 80.4± 0.01 4.7 ± 0.07 19.6 ± 0.1 16.9 ± 0.1 0.997 1.981 (18 yr)

TABLE 11 Comparison of time resolved fluorescence lifetimes from 42 yrold non-diabetic and 42 yr old type II diabetic human donor lenses byexcitation at 435 nm and emission maxima between 500-510 nm Type ofLifetimes, 

 (ns) and Durbin - donor Relative contributions at time zero, A (%)Watson lens A₁

A₂

A₃

 value parameter Non- — — — — — — — — diabetic (42 yr) Diabetic — — 84.1± 2.2 4.2 ± 0.77 15.9 ± 3.2 17.1 ± 0.5 1.073 1.74 (42 yr)

TABLE 12 Comparison of time resolved fluorescence lifetimes from donorlenses of 56 yr old non-diabetic and 56 yr old with diabetic retinopathyby excitation at 435 nm and emission maxima between 500-510 nm Type ofLifetimes, 

 (ns) and Durbin - donor Relative contributions at time zero, A (%)Watson lens A₁

A₂

A₃

 value parameter Non- — — 100 4.5 ± 0.17 — — 1.07 1.79 diabetic (56 yr)Diabetic — — 51.4 ± 0.8 4.6 ± 0.11 48.6 ± 0.2 17 ± 0.17 1.073 1.74 (56yr)

TABLE 13 Comparison of time resolved fluorescence lifetimes from donorlenses of 64 yr old non-diabetic and 67 yr old with cataract byexcitation at 435 nm and emission maxima between 500-510 nm Type ofLifetimes, 

 (ns) and Durbin - donor Relative contributions at time zero, A (%)Watson lens A₁

A₂

A₃

 value parameter Non- — — 100 4.76 ± 0.1 — — 0.992 1.86 diabetic (64 yr)Diabetic 39 ± 1.9 1.4 ± 0.2 41.9 ± 0.5 4.5 ± 0.3 20.1 ± 1.2 15.6 ± 0.91.073 1.94 (67 yr)

1. A method for characterizing advanced glycation end-products (AGEs) inan ocular cell or tissue sample, the method comprising performing afluorescent measurement of the cell or tissue to quantify the AGEs inthe cell or tissue, and comparing the quantified fluorescent measurementof the AGEs to a standardized AGE fluorescent measurement.
 2. The methodfor characterizing AGEs of claim 1, wherein the ocular sample is fromthe lens.
 3. The method of claim 1 used for early diagnosis of ahyperglycemic microenvironment, wherein the fluorescent measurements areexcited at wavelengths between 400-600 nm.
 4. The method of claim 1,further defined as detecting changes in AGEs over time and in responseto treatments for a condition or disease.
 5. The method of claim 1,wherein the AGE is formed on a crystallin protein.
 6. The method ofclaim 1, wherein the fluorescent measurement is a fluorescent lifetimedetection.
 7. The method of claim 1, wherein the fluorescent measurementis a time resolved fluorescence measurement.
 8. The method of claim 4,wherein the disease is diabetes mellitus.
 9. An apparatus for detectingadvanced glycation endproducts (AGE) in a cell or tissue, the apparatuscomprising: means for focusing an excitation light beam on the cell ortissue; means for detecting a fluorescent light emitted from the cell ortissue in one or more wavebands, wherein the detecting means produces anintensity of fluorescence from the cell or tissue; means for calculatingthe intensity of fluorescence for each waveband, wherein the calculatingmeans detects the AGEs in the cell or tissue in the form of an intensitymap; and means for distinguishing normal from pathological cell ortissue based on differences in the fluorescence intensity maps.
 10. Amethod for detecting and characterizing fluorophores in an ocularprotein, cell or tissue, the method comprising: (a) focusing anexcitation light beam on the ocular protein, cell or tissue; (b)detecting a fluorescent light emitted from the ocular protein, cell ortissue in a one or more wavebands, by an intensity of fluorescence fromthe ocular protein, cell or tissue; (c) calculating the intensity offluorescence for each waveband, wherein the calculation detects thefluorophore in the ocular protein, cell or tissue; (d) measuringfluorescence decay with time after a short excitation pulse from theocular protein, cell or tissue in the specific waveband; and (e)calculating the fluorescence lifetimes from the fluorescent decay curvesusing global 1-4 three exponential fitting analysis.
 11. The method ofclaim 10, wherein the fitting analysis is calculated based on goodautocorrelation function around zero, weighted number of residualsrandomly distributed between +3 and −3, reduced chi square valuesbetween 0.9-1.1 and Durbin-Watson parameters of greater than or equal to1.6, 1.7 and 1.8 for one, two and three exponential decays respectively.12. An apparatus for detecting fluorophores in an ocular protein, cellor tissue, the apparatus comprising: means for focusing an excitationlight beam on the ocular protein, cell or tissue; means for detecting afluorescent light emitted from the cell or tissue in a one or morewavebands, wherein said detection means is operable to produce anintensity of fluorescence from the protein, cell or tissue; means forcalculating the intensity of fluorescence for each waveband, wherein thecalculation means discerns quantifying the lifetimes of the differentfluorophores formed in the protein, cell or tissue.
 13. An in vitrodiagnostic test using the method of claim 10 for distinguishing thenormal samples from pathological samples, further defined as based onthe fluorescence intensity, decay and change in lifetime measurements atspecific excitation wavelengths between 280-600 nm, and the damage tothe proteins in the tissue is confirmed based on mass spectrometry andX-ray scattering.